1. Field of the Invention
The present invention is directed to DNA molecules encoding imidazoline receptive polypeptides, preferably encoding human imidazoline receptive polypeptides, that can be used as an imidazoline receptor (abbreviated IR). In addition, transcript(s) and protein sequences are predicted from the DNA clones. The invention is also directed to a genomic DNA clone designated as JEP-1A. The cDNA clones according to the invention comprise cDNA homologous to portion(s) of this genomic clone; including 5A-1 cDNA, cloned by the inventors that established the open-reading frame for translation of mRNA from the gene, and established the immunoreactive properties of its polypeptide sequence in an expression systems. Also, the invention relates to cDNA clone EST04033, which is another clone identified to contain cDNA sequences from the JEP-1A gene, and of which the 5A-1 is a part, that encodes an active fragment of the IR polypeptide in transfection assays, and the protein sequences thereof. The invention also relates to methods for producing such genomic and cDNA clones, methods for expressing the IR protein and fragments, and uses thereof.
2. Description of Related Art
It is believed that brainstem imidazoline receptors possess binding site(s) for therapeutically relevant imidazoline compounds, such as clonidine and idazoxan. These drugs represent the first generation of ligands discovered for the binding site(s) of imidazoline receptors. However, clonidine and idazoxan were developed based on their high affinity for α2-adrenergic receptors. Second generation ligands, such as moxonidine, possess somewhat improved selectivity for IR over α2-adrenergic receptors, but more selective compounds for IR are needed.
An imidazoline receptor clone is of particular interest because of its potential utility in identifying novel pharmaceutical agents having greater potency and/or more selectivity than currently available ligands have for imidazoline receptors. Recent technological advances permit pharmaceutical companies to use combinatorial chemistry techniques to rapidly screen a cloned receptor for ligands (drugs) binding thereto. Thus, a cloned imidazoline receptor would be of significant value to a drug discovery program.
Until now, the molecular nature of imidazoline receptors remains unknown. For instance, no amino acid sequence data for a novel IR, e.g., by N-terminal sequencing, has been reported. Three different techniques have been described in the literature by three different laboratories to visualize imidazoline-selective binding proteins (imidazoline receptor candidates) using gel electrophoresis. Some important consistencies have emerged from these results despite the diversity of the techniques employed. On the other hand, multiple protein bands have been identified, which suggests heterogeneity amongst imidazoline receptors. These reports are discussed below.
Some of the abbreviations used hereinbelow, have the following meanings:
 α2AR     Alpha-2 adrenoceptorBACBovine adrenal chromaffinECLEnhanced chemiluminescence (protein detectionprocedure)ESTExpressed Sequence Tag (a one-pass cDNAdocumentation without identification)I-siteAny imidazoline-receptive binding site (e.g.,encoded on IR)IR1Imidazoline receptor subtype1IR-AbImidazoline receptor antibodyI2SiteImidazoline binding subtype2kDaKilodaltons (molecular size)MAOmonoamine oxidaseMWmolecular weightNRLEuropean abbreviation for RVLM (see below)PC-12Phaeochromocytoma-12 cells125PIC[125I]p-iodoclonidinePKCProtein Kinase CRVLMRostral Ventrolateral Medulla in brainstemSDSsodium dodecyl sulfate gel electrophoresis
Reis et al. [Wang et al., Mol. Pharm., 42: 792-801 (1992); Wang et al., Mol. Pharm., 43: 509-515 (1993)] were the first to characterize an imidazoline-selective binding protein and to demonstrate it as having MW=70 kDa. This was accomplished using bovine cells (BAC), which lack an α2AR [Powis & Baker, Mol. Pharm., 29:134-141 (1986)]. The 70 kDa imidazoline-selective protein in those studies had high affinities for both idazoxan and p-aminoclonidine affinity chromatography columns and was eluted by another imidazoline compound (phentolamine). Unfortunately, those investigators failed to isolate sufficient 70 kDa protein to determine its other biochemical properties. To date, no one has reported the complete purification of an imidazoline receptor protein. Likewise, no amino acid sequences have been reported for IR.
Their 70 kDa protein was used by Reis and co-workers to raise “I-site binding antiserum”, designated herein as Reis antiserum. The term “I-site” refers to the imidazoline binding site, presumably defined within the imidazoline receptor protein. Reis antiserum was prepared by injecting the purified protein into rabbits [Wang et al, 1992]. The first immunization was done subcutaneously with the protein antigen (10 μg) emulsified in an equal volume of complete Freund's adjuvant, and the next three booster shots were given at 15-day intervals with incomplete Freund's adjuvant. The polyclonal antiserum has been mostly characterized by immunoblotting, but radioimmunoassays (RIA) and/or conjugated assay procedures, i.e., ELISA assays, are also conceivable [see “Radioimmunoassay of Gut Regulatory Peptides: Methods in Laboratory Medicine,” Vol. 2, chapters 1 and 2, Praeger Scientific Press, 1982].
The present inventors and others [Escriba et al., Neurosci. Lett. 178: 81-84 (1994)] have characterized the Reis antiserum in several respects. For instance, the present inventors have discovered that human platelet immunoreactivity with Reis antiserum is mainly confined to a single protein band of MW≈33 kDa, although a trace band at ≈85 kDa was also observed. The ≈33 and ≈85 kDa bands were enriched in plasma membrane fractions as expected for an imidazoline receptor. Furthermore, the intensity of the =33 kDa band was found to be positively correlated with non-adrenergic 125PIC Bmax values at platelet IR1 sites in samples from the same subjects, with an almost one-to-one slope factor. In addition, the nonadrenergic 125PIC binding sites on platelets were discovered by the present inventors to have the same rank order of affinities as IR1 binding sites in brainstem [Piletz and Sletten, J. Pharm. & Exper. Therap., 267: 1493-1502 (1993)]. The platelet≈33 kDa band may also be a product of a larger protein, since in human megakaryoblastoma cells, which are capable of forming platelets in tissue cultures, an ≈85 kDa immunoreactive band was found to predominate.
Immunoreactivity with Reis antiserum does not appear to be directed against human α2AR and/or MAO A/B. This is a significant point because α2AR and MAO A/B have previously been cloned and also bind to imidazolines. The present inventors have obtained selective antibodies and recombinant preparations for α2AR and MAO A/B, and these proteins do not correspond to the ≈33, 70, or 85 kDa putative IR1 bands. Thus, there is substantial evidence that, at least in human platelets, the Reis antiserum is IR1 selective.
Another antiserum was raised by Drs. Dontenwill and Bousquet in France [Greney et al., Europ. J. Pharmacol., 265: R1-R2 (1994); Greney et al., Neurochem. Int., 25: 183-191 (199.4); Bennai et al., Annals NY Acad. Sci., 763:140-148 (1995)] against polyclonal antibodies for idazoxan (designated Dontenwill antiserum). This anti-idiotypic antiserum inhibits 3H-clonidine but not 3H-rauwolscine (α2-selective) binding sites in the brainstem, suggesting it also interacts with IR1 [Bennai et al., 1995]. As shown in FIG. 1, human RVLM (same as NRL) membrane fractions displayed bands of ≈41 and 44 kDa, as detected by the present inventors using this anti-idiotypic antiserum.
The present inventors have found that the bands of MW≈41 and 44 kDa detected by Dontenwill antiserum may be derived from an ≈85 kDa precursor protein, similar to that occurring in platelet precursor cells. An 85 kDa immunoreactive protein is obtained in fresh rat brain membranes only when a cocktail of 11 protease inhibitors is used. Also, as shown in FIG. 1, it is found that Reis antiserum detects the ≈41 and 44 kDa bands in human brain when fewer protease inhibitors are used. Additionally, the Dontenwill antiserum weakly detects a platelet ≈33 kDa band. Thus, the present inventors have hypothesized that the ≈41 and 44 kDa immunoreactive proteins may be alternative breakdown products of an ≈85 kDa protein, as opposed to the platelet≈33 kDa breakdown product.
In summary, the main conclusion from the above results is that, despite vastly different origins, the Reis and Dontenwill antisera both detect identical bands in human platelets, RVLM, and hippocampus.
Using yet another technique, a photoaffinity imidazoline ligand, 125AZIPI, has also been developed to preferentially label I2-imidazoline binding sites [Lanier et al., J. Biol. Chem., 268: 16047-16051 (1993)]. The 125AZIPI photoaffinity ligand was used to visualize ≈55 kDa and ≈61 kDa binding proteins from rat liver and brain. It is believed that the ≈61 kDa protein is probably MAO, in agreement with other findings [Tesson et al., J. Biol. Chem., 270: 9856-9861 (1995)] showing that MAO proteins bind certain imidazoline compounds. The different molecular weights between these bands and those detected immunologically by the present inventors is one of many pieces of evidence that distinguishes IR1 from I2 sites.
To the inventors' knowledge and as described herein, we are first to clone the gene, cDNAs and fragments thereof encoding a protein with the immunological and ligand binding properties expected of an IR. on this basis, we are first to identify the nucleotide sequences of DNA molecules encoding an imidazoline receptor and active fragments thereof, and the first to determine the amino acid sequence of an imidazoline receptor and active fragments thereof. The polypeptides described herein are clearly distinct from α2AR or MAO A/B proteins.